Cognitive impairment is associated with a number of diseases and conditions, including Alzheimer's disease, stroke, neural disorders and mental retardation. Drugs which enhance memory and facilitate learning by enhancing LTP would be useful in treating individuals with these conditions. In addition, drugs of this type would be valuable in enhancing learning in normal individuals.

SUMMARY OF THE INVENTION Disclosed herein are novel methods of enhancing cognitive functions in mammals and methods of treating an individual with cognitive dysfunctions using Breflate (1) and analogs of Reflate. Also disclosed are novel compounds which can be used for these purposes.

One embodiment of the present invention is a method of treating a mammal with a cognitive dysfunction. The method comprises administering a therapeutically effective dose of a compound represented by Structural Formula (I), or physiologically acceptable salts thereof.

Alternatively, the method comprises administering a therapeutically effective dose of a compound represented by Structural Formula (II), or physiologically acceptable salts thereof.

Another embodiment of the present invention is a method of enhancing cognitive functions, for example, memory and learning, in a mammal. The method comprises administering a memory enhancing amount of a compound represented by Structural Formula (I), or physiologically acceptable salts thereof. Alternatively, the method comprises administering an effective cognitive enhancing amount of a compound represented by Structural Formula (II), or physiologically acceptable salts thereof.

Structural Formula (I) is shown below: X1 is selected from the group consisting of: X2 is selected from the group consisting of: X3 is a lower alkyl group, preferably methyl.

R1 is selected from the group consisting of:

R2 and R3 are independently selected from the group consisting of -H, an aliphatic group, a substituted aliphatic group, an aryl group, a substituted aryl group, monosaccharides, NH2-CHR6-CO-, -OH, -O(aliphatic or substituted aliphatic group) and -O-(aryl or substituted aryl group).

R4 and R5 are independently selected from the group consisting of -H, aliphatic groups, aromatic groups, substituted aliphatic groups and substituted aromatic groups.

R6 is the side chain of an amino acid.

Structural Formula (II) is shown below:

One of R1l and R12 is -H and the other of R1l and R12 is a substituent group having 1 to 12 carbon atoms containing a basic nitrogen atom or a quaternary ammonium group, or a salt thereof. This substituent group can more preferably have 1 to 8 carbon atoms, most preferably 1 to 4 carbon atoms.

Preferably, one of R1l and R12 is a group of the formula -COR13, wherein R13 is selected from the group consisting of an aliphatic group containing 1 to 12 carbon atoms, more preferably 1 to 8 carbon atoms, most preferably 1 to 4 carbon atoms; an aryl aliphatic group containing 1 to 12 carbon atoms, more preferably 1 to 9 carbon atoms; and a heterocyclic group containing 4 to 6 carbon atoms, each of said groups containing a basic nitrogen atom or a quaternary ammonium group. The aryl aliphatic group can be an arylalkyl group wherein the aryl group can be a benzene ring, and the alkyl group contains 1 to 3 carbon atoms. The aryl aliphatic group can have a basic nitrogen atom or quaternary ammonium group on the aliphatic moiety attached to the ester group of the compound, or on a side chain of the aryl moiety of the aryl aliphatic group.

One of R11 and R12 can be a group of the formula:

wherein n is an integer of 1 to 3, and R14 and R15 are independently or identically selected from the group consisting of hydrogen and an alkyl group having from 1 to 3 carbon atoms, or wherein R14 and R15 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring having 4 to 6 carbon atoms wherein 1 to 2 of said carbon atoms can optionally be replaced by heteroatoms. These additional heteroatoms can be selected from N, S, and 0. If the heterocyclic ring contains only one nitrogen atom, the number of carbon atoms would be 4 to 6. If the heterocyclic ring contains two heteroatoms, one of which is nitrogen, the number of carbon atoms is 3 to 5. A 7 membered heterocyclic ring can contain 2 or 3 heteroatoms, of which one would be nitrogen; with 2 heteroatoms, the number of carbon atoms would be 5, and with 3 heteroatoms, the number of carbon atoms would be 4.

Preferably, R13 is a group of the formula -CH2N(CH3)2, or a residue obtained by removing the carboxyl group of an amino acid. Examples include glycine, alanine,

leucine, isoleucine, valine, phenylalanine, proline, lysine and arginine.

Suitable heterocyclic rings having 1 to 2 heteroatoms include a piperazine ring, a methylpiperazine ring, a morpholine ring, a methylmorpholine ring, a perhydrothiazine ring, a methylperhydrothiazine ring, a pyrazoline ring, an isoxazoline ring, an oxazoline ring, a 1,2-oxazoline ring, a perhydropyridazine ring, a perhydrotriazine ring, a perhydrooxadiazine ring, a perhydrooxadiazine ring, an imidazoline ring, a thiazoline ring, an isothiazoline ring, a 1,2,4- oxadiazoline ring, a 1,2,5-oxadiazoline ring, 1,2,3- triazole, a 1,2,4-triazole an azepine ring, a perhydroazepine ring, a 1,2,4-diazepine ring, a perhydrothiazepine ring, a perhydrooxazepine ring, and a perhydrothiazepine ring.

Yet another embodiment of the present invention is a compound represented by Structural Formula (I) and physiologically acceptable salts thereof. X1, X2, X3 and R1-R6 are as described above for Structural Formula (I).

Breflate (1) and the analogs of Breflate disclosed herein can enhance LTP in vivo and memory and learning in laboratory rats and mice at low doses. In addition, Breflate and the analogs of Breflate disclosed herein are highly soluble in physiological solution. Thus, they are likely to be useful as drugs for treating diseases associated with cognitive impairment and for enhancing learning in cognitively normal mammals, including humans.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graph showing the effect of various doses of Breflate administered two hours prior to training on an inhibitory-avoidance task in laboratory mice, measured by the latency time in seconds.

Figure 2 is a graph showing the effect of various doses of Breflate administered immediately post-training

on an inhibitory-avoidance task in laboratory rats, measured by the latency time in seconds.

Figure 3 is a graph showing the effect of 3.0 mg Breflate per kg body weight administered to laboratory mice at different times relative to training on an inhibitory-avoidance task, measured by the latency time in seconds.

Figure 4 is a graph showing the effect of various concentrations of Breflate on learning when administered to laboratory mice in the Y-maze discrimination test, as measured by the number of errors made by the mice during the test phase.

DETAILED DESCRIPTION OF THE INVENTION Disclosed herein are methods of treating a mammal with a cognitive dysfunction. As used herein, "cognitive dysfunction" refers to conditions and/or diseases which result in cognitive impairment, for example, loss of memory or a greater difficulty in learning than prior to the onset of the disease or condition. Conditions and diseases which cause cognitive impairment include Alzheimer's disease, Down's Syndrome, senility, stroke, mental retardation and other neural disorders.

Also disclosed herein are methods of enhancing cognitive functions, e.g., learning and memory in a mammal. "Enhancing cognitive functions" refers to, for example, causing a mammal without cognitive dysfunction, e.g., a normal individual, to learn quicker with the treatment than without the treatment, or to have improved memory as a result of the treatment than without the treatment.

"Mammals" includes humans, laboratory animals (e.g., rats, mice, guinea pigs and the like), farm animals (e.g., horses, pigs, cows and the like) and veterinary animals (e.g. dogs, cats and the like).

An aliphatic group includes straight chained or branched lower alkyl groups (e.g., C1 to about C8

hydrocarbons), optionally with one or more units of unsaturation and/or with one to two heteroatoms (e.g., sulfur, oxygen and nitrogen) in the hydrocarbon chain.

Thus, lower alkenes and lower alkynes alkynes (e.g., C2 to about C8 hydrocarbons) are included within the meaning of the term "aliphatic group", as it is used herein. The term "aliphatic group" also includes C3 to about C8 cycloalkyl groups, optionally with one or more units of unsaturation and/or with one to three heteroatoms in the ring. Thus, heterocyclic groups such as oxazolidines are included within the meaning of the term "aliphatic group", as it is used herein. A "substituted aliphatic group" can have one or more substituents, e.g., an aryl group (including a carbocyclic aryl group or a heteroaryl group), a substituted aryl group, -O-(aliphatic group or aryl group), -O-(substituted aliphatic group or substituted aryl group), acyl, -CHO, -CO-(aliphatic or substituted aliphatic group), -CO-(aryl or substituted aryl), -COO- (aliphatic or substituted aliphatic group), -NH-(acyl), -O-(acyl), benzyl, substituted benzyl, halogenated lower alkyl (e.g. trifluoromethyl and trichloromethyl), fluoro, chloro, bromo, iodo, cyano, nitro, -SH, -S-(aliphatic or substituted aliphatic group), -S-(aryl or substituted aryl), -S-(acyl) and the like. Acyl is -CO-(aliphatic, substituted aliphatic, aryl or substituted aryl group).

Suitable aromatic groups (or aryl groups) include carbocyclic aromatic groups, such as phenyl and naphthyl, and heteroaryl groups, such as monocyclic or polycyclic aromatic groups containing one or more heteroatoms such as oxygen, nitrogen or sulfur. Examples of suitable monocyclic heteroaryl groups include imidazolyl, thienyl, pyridyl, furanyl, oxazolyl, pyrollyl, pyrimidinyl, furanyl, pyrazolyl, pyrrolyl, thiazolyl and the like. A polycyclic heteroaryl group includes fused structures such as quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzothiazolyl, benzothiophenyl,

benzofuranyl and benzopyranyl. Suitable substituents include aliphatic groups, substituted aliphatic groups and those substituents described above for aliphatic groups.

As used herein, a "monosaccharide" is a chiral polyhydroxy aldehyde in a cyclic hemiacetal form (referred to as an "aldose") or a chiral polyhydroxy ketone in a cyclic hemiketal form (referred to as a "ketose"). Examples of suitable polyhydroxy aldehydes include aldotrioses, aldotetroses (e.g. D- and L-erythrose and threose), aldopentoses (e.g. D- and L-arabinose, xylose, ribose, and lyxose), aldohexoses (e.g. D- and L-glucose, allose, altrose, mannose, gulose, idose, galactose and talose), aldoheptoses, aldo-octoses and aldononoses. The polyhydroxy aldehyde can be in a furanose form (for aldoses having four carbons or more) or pyranose form (for aldoses having five carbon atoms or more).

Suitable polyhydroxy ketones include pentuloses, hexuloses (e.g. D-fructose, D-sorbose, D-psicose and D-tagatose), heptuloses, octuloses and nonuloses. The polyhydroxy ketone can be in a furanose form (for ketoses having five carbons or more) or pyranose form (for ketoses having six or more carbon atoms).

When R2 or R3 is a monosaccharide, R2 or R3 is connected to the designated nitrogen atom in Structural Formula (I) by a single covalent bond between the designated nitrogen atom and the anomeric carbon of the monosaccharide.

Suitable amino acids include naturally occurring amino acids. Also included are non-naturally occurring amino acids in which the side chain is an aliphatic group, a substituted aliphatic group, an aromatic group or a substituted aromatic group, as defined above. When R2 or R3 is an amino acid, R2 or R3 is connected to the designated nitrogen atom in Structural Formula (I) by an

amide bond between the designated nitrogen atom and the C-terminus of the amino acid.

A "therapeutically effective dose" is the quantity of compound, which, when administered to a cognitively impaired mammal, results in the mammal having improved cognitive function than in its absence. An "effective cognitive enhancing amount" is the quantity of compound, which, when administered to a cognitively unimpaired mammal, results in the mammal having improved cognitive function than in its absence. Improved cognitive function includes, for example, improved memory and learning ability. Cognitive function in individuals can be measured by standard tests, for example, in humans by, for example, the Information-Memory-Concentration subtest of the Blessed Dementia Rating Scale or the Mini Mental Status Exam (Corey-Bloom et al., Neurology, 45:211 (1995)). Cognitive function in animals can be measured by tests such the inhibition avoidance test described in Examples 2-4 and the Y-maze test described in Example 5.

Therapeutically effective and effective cognitive enhancing doses range from about 0.005 mg/kg body weight to about 5 mg/kg body weight, preferably from about 0.01 mg/kg body weight to about 0.5 mg/kg body weight.

Breflate can be prepared by synthetic procedures disclosed in WO 96/00726 by Malspeis et al., the entire teachings of which are incorporated herein by reference.

Analogs of Breflate can be prepared by suitable modifications of these synthetic procedures.

The compound can be administered orally, for example, in capsules, suspensions or tablets.

Preferably, the compound is administered parenterally.

Modes of parenteral administration which can be used include systemic administration, such as by intramuscular, intravenous, subcutaneous, or intraperitoneal injection.

The compound can be administered to the individual in conjunction with an acceptable pharmaceutical carrier.

Suitable pharmaceutical carriers may contain inert ingredients which do not interact with the compound.

Standard pharmaceutical formulation techniques may be employed such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9k mg/ml benzyl alcohol), phosphate- buffered saline, Hank's solution, Ringer's-lactate and the like. Methods for encapsulating compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art (Baker, et al., "Controlled Release of Biological Active Agents", John Wiley and Sons, 1986).

The invention is illustrated by the following examples, which are not intended to be limiting in any way.

EXAMPLE 1 - INDUCTION OF LONG-TERM POTENTIATION BY BREFLATE IN VIVO Transverse slices (500 ym) were cut from the hippocampus of 4-8 weeks old rats, submerged and continuously perfused at 2-3ml/min with oxygenated artificial cerebrospinal fluid at 300C. Excitatory post- synaptic potentials were elicited ever 5 sec by stimulation of Schaffer collateral/commissural fibers with bipolar tungsten stimulating electrodes, and extracellular field potentials were recorded from the CA1 pyramidal cell layer before and after Breflate (0.01- 10yg/ml) application with glass micro electrodes filled with artificial cerebrospinal fluid. A stock solution of Breflate in dilute aqueous HCl (1 mg/ml) was diluted with oxygenated artificial cerebrospinal fluid and applied to the hippocampal slices at 2-3 ml/min for 2 to 10 minutes.

To evaluate the effect of the drug, the population spike amplitude was monitored before and after drug application.

In several slices, it was found that Breflate increased the amplitude of population spikes (PS) in CA1 area immediately or 10-20 minutes after the drug application. In all cases, tetanus induced further potentiation.

EXAMPLE 2 - ENHANCEMENT OF LEARNING IN MICE BY TREATMENT WITH BREFLATE, MEASURED BY THE INHIBITORY-AVOIDANCE TASK TEST Tests were conducted to examine the effects of Breflate on memory in mice. Mice received an injection of Breflate (0.1 to 10 mg/kg, i.p.) or saline two hours before training in the inhibitory-avoidance task.

The subjects were 60-day-old (23-28 g) male CFW mice (Charles River Laboratories). The mice were acclimatized to laboratory conditions for 1 week before the training began. They were housed six to a cage and maintained on a 12-h light-dark cycle (lights on a 7:00 A.M.) with food and water freely available. The experiments were conducted between 9:00 A.M. and 2:00 P.M.

Mice were first trained on a trough-shaped step- through inhibitory avoidance apparatus. On the training trial, each mouse was placed in the lighted compartment, facing away from the dark compartment and a door leading to dark compartment was opened. When the mouse stepped through the door into the dark compartment the door was closed and the foot shock (0.7 mA, 60 Hz, 2 s) was delivered. On the retention test 24 h later, the mouse was placed in the lighted compartment as on the training session and the step-through latency (maximum of 300 s) was recorded.

The results are shown in the attached Figure 1. The average observed retention latencies of the 0.1 and 3.0 mg/kg doses were significantly greater than that of the

saline controls, indicating enhancement memory. It is not clear at this point, why no effect at the 0.3 and 1.0 mg/kg doses was found.

EXAMPLE 3 - ENHANCEMENT OF LEARNING IN RATS BY TREATMENT WITH BREFLATE, MEASURED BY THE INHIBITORY-AVOIDANCE TASK TEST The subjects were 50-day-old male Sprague-Dawley rats (Charles River Labs.) 180-200 g on arrival. The rats were individually housed with continuous access to food and water, maintained on a 12-h light-dark cycle (lights on at 07.00) and acclimatized to laboratory conditions for one week before behavioral studies. The experiments were conducted between 09.00 and 15.00 h.

The rats were trained on a trough-shaped inhibitory avoidance (1A) apparatus consisting of two compartments separated by a sliding door that opens by retracting into the floor. The starting compartment was illuminating by a tensor lamp which provided the only illumination in the testing room. On the training trail, the rat was placed in he lighted compartment, facing the closed door. When the rat turned around, the door leading to the dark compartment was opened. When the rat stepped through the door into the dark compartment the door was closed, a foot shock (0.35 mA, 60 Hz, 0.7s) was delivered, and the response latency was recorded.

The rat was then immediately removed from the apparatus and injected i.p. with a solution of Breflate (0.01, 0.1, or 1.0 mg/kg,i.p.) or saline. Retention was tested 48 hours later. On the retention test, the rat was placed in the lighted compartment as on the training session and the step-through latency (maximum of 500s) was recorded.

The results are shown in Figure 2. The retention latencies of all three drug injected groups were higher than those of controls, indicating enhanced memory,

although this effect was significant for the 0.01 and 1.0 doses.

Initial findings with Breflate in both mice (Fig. 1) and rats (Fig. 2) indicate that the dose-response function for memory enhancement by the drug is bi-modal.

In both species, significant enhancement of memory has been found at two dose levels, but not at levels between those two.

EXAMPLE 4 - EFFECT OF THE TIME OF ADMINISTRATION OF BREFLATE RELATIVE TO TRAINING ON LEARNING IN MICE, AS DETERMINED BY THE INHIBITORY- AVOIDANCE TASK TEST A time course study using the inhibitory-avoidance test described in Examples 2 and 3 was conducted with laboratory mice. Mice were injected with Breflate or vehicle at one of the following time points: 2 weeks pre- training; 1 week pre-training; 2 hours pre-training; immediately post-training; 2 hours post-training; or 6 hours post-training. Retention was tested 48 hours later. The results are shown in Figure 3. Breflate significantly enhanced retention when administered 2 hours pre-training, but not when administered 1 or 2 weeks pre-training. It is clear from the results that the drug did not affect memory when given 2 weeks pre- training. A significant enhancement was observed in the 2 hour pre-training group, in the immediately post- training group and in the six hour post-training group.

However, a potential enhancing effect of the drug in the 1 week pre-training group may have been obscured by higher than normal control performance. The same explanation may account for the lack of effect of Breflate in this experiment in the 2 hour post-training group.

EXAMPLE 5 - LEARNING ENHANCEMENT BY BREFLATE IN LABORATORY MICE, AS MEASURE BY THE Y-MAZE DISCRIMINATION TEST In the Y-Maze Discrimination Task (YMD), animals indicate memory of foot shock escape training by persisting in choosing an alley of the Y-maze that previously allowed escape from foot shock (even though entrances of that alley were punished with foot shock in the retention test trials).

The Y-maze apparatus consists of a lucite chamber with three arms, each approximately one foot long. The floors of the arms have two plates separated by less than a centimeter connected to a positive and a negative terminal so as to deliver a 0.35 mA shock to the foot of the test animal. The Y-maze is used to test the animal's capacity to remember which of the two arms delivered a shock during the preliminary learning phase. Therefore, during the testing phase, the safe arm is reversed from the one that was safe during the learning phase. The more the animal remembers from the learning phase, the more errors it will make during the test phase.

Consequently, a high error rate indicates learning of the task.

The training phase of the Y-maze discrimination reversal task was as follows. Animals were place in a three arm lucite Y-maze apparatus, with each arm separated by a sliding door. The left arm was illuminated during testing as during training, while the center arm and the right arm remained dark. Animals were placed in the darkened center (start arm) arm, (at the base of the Y); after 10 second, a foot shock (0.35 mA, 60 Hz) was delivered to the start arm and to the intersection of all three arms, but not to the illuminated arm until the animal entered the lighted arm.

Mice that failed to enter the lighted arm within 60 seconds were removed and placed again into the start arm.

Those that had fully entered the illuminated arm were

lifted and returned to the start arm. The interval between training trials was 40 seconds. The final stage of training required that, when given the choice between the lighted and dark arms, the mice successfully chose the lighted arm three consecutive times (the criterion).

All mice were then subjected to a forced entry into the dark arm where they received a five second foot shock to ensure that all animals experienced foot shock in the dark arm. Dosing with Breflate solution (3 mg/kg body weight) or saline was given immediately post training.

Testing was conducted 48 hours later, but with the dark arm as the safe arm and the illuminated arm providing a foot shock. The number of errors in the trials was recorded. As shown by the data in Figure 4, administration of Breflate at a dosage of 3.0 mg/kg body weight administered immediately post training revealed a statistically significant memory enhancement.

EQUIVALENTS Those skilled in the art will be able to recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.